David S Peal et al. (2001) International C. elegans Meeting "Chemical and Genetic Inhibition of Sulfation in C. elegans"

David S Peal, Laura L Kiessling

[International C. elegans Meeting, 2001]

In order to examine the process of sulfation in C. elegans, sulfation was inhibited chemically using sodium chlorate, and genetically using the process of RNA-mediated interference (RNAi). Sodium chlorate inhibition during early larval stages resulted in a dose-dependant developmental delay. BLAST searches of characterized sulfotransferases against the worm genome resulted in the identification of 4 putative sulfotransferases: C34F6.4 and F08B4.6 (previously identified: [1] and [2]), F40H3.5, and Y34B4A.e. RNAi of the putative N-deacetylase/N-sulfotransferase F08B4.6 resulted in "stacking" of eggs in the gonad, along with eggs laid at the 2- and 4-celled stage. RNAi of the putative hexuronic 2-O sulfotransferase C34F6.4 resulted in a shortened, bulbous gonad. These initial results indicate that sulfation may be important during development of C. elegans. [1] Shworak, NW, Liu, J, Fritze, LMS, Schwartz, JJ, Zhang, L, Logeart, D, Rosenberg, RD. JBC 272: 28008-19 (1997). [2] Kobayashi, M, Sugumaran, G, Liu, J, Shworak, NW, Silbert, JE, Rosenberg, RD. JBC 274: 10474-80 (1999).

Martin Gutternigg et al. (2006) European Worm Meeting "Glycosidases of Caenorhabditis elegans involved in N-glycan processing"

Gunter Lochnit, Iain B. H. Wilson, Petra Geier, Ramona Ranftl, Martin Gutternigg, Katharina Paschinger, Matthias Hackl, Ute Stemmer, Dorothea Lubich, Verena Jantsch

[European Worm Meeting, 2006]

Martin Gutternigg, Dorothea Lubich, Matthias Hackl, Katharina Paschinger, Ute Stemmer, Verena Jantsch1, Gnter Lochnit2, Ramona Ranftl, Petra Geier and Iain B. H. Wilson. Recent data indicates that in addition to the Golgi ?-mannosidases, the model nematode Caenorhabditis elegans also possesses, like insects, an N-acetylhexosaminidase activity putatively involved in N-glycan processing in the Golgi. The presence of such an activity is invoked, not just on the basis of the detected enzyme activity, but also to explain the absence of terminal N-acetylglucosamine residues on structures which require the prior action of N-acetylglucosaminyltransferase I during their biosynthesis. In order to understand the genetic basis for these activities, we have cloned cDNAs encoding members of both glycohydrolase families 20 and 38 from the worm. The encoded glycosidases were expressed in the yeast Pichia pastoris as soluble forms lacking putative cytoplasmic and transmembrane domains. Four glycohydrolase family 20 members were shown to cleave p-nitrophenyl-?-N-acetylglucosaminide and/or p-nitrophenyl-?-N-acetylgalactosaminide, but showed contrasting specificities with regard to N-glycan substrates. On the other hand, one glycohydrolase family 38 member was shown to be active using p-nitrophenyl-?-mannoside as a substrate and, in addition, had mannosidase II activity. Analysis of the glycans of the relevant mutant showed large-scale changes in the N-glycosylation spectrum. These, therefore, are the first data on the activity of Caenorhabditis glycosidases towards N-glycan substrates and should aid the further elucidation of N-glycan processing in this organism.

Gary Schindelman et al. (2002) West Coast Worm Meeting "Characterization of the sperm transfer step of male mating behavior of Caenorhabditis elegans ."

Allyson J Whittaker, Gary Schindelman, Paul W Sternberg, Shahla Gharib

[West Coast Worm Meeting, 2002]

C. elegans male mating behavior involves the proper execution of a series of sub-behaviors culminating in the transfer of sperm to the hermaphrodite (Liu, KS and Sternberg, PW. 1995. Neuron 14:79-89). These sub-behaviors are: Response to hermaphrodite, backing, turning, vulval location, spicule insertion and sperm transfer. We are analyzing the genetic control of this stereotyped behavior as it may provide insight into sensory perception and nervous system function.

ZHANG, Gaotian et al. (2015) International Worm Meeting "Characterization of nematode-infecting microsporidia and natural variation in sensitivity of Caenorhabditis nematodes to microsporidia."

Felix, Marie-Anne, Zhang, Gaotian

[International Worm Meeting, 2015]

The microsporidian Nematocida parisii is the first characterized intracellular pathogen of Caenorhabditis elegans; and a second Nematocida species, called N. sp. 1, was also reported from an Indian C. briggsae isolate (Troemel et al. 2008). In our lab we further established a collection of wild rhabditid nematodes suspected with microsporidian infection. Here, we characterize molecularly these wild microsporidia and study their specificity of infection.First, the potentially infected rhabditid isolates were characterized using newly designed PCR primers specific for 18S and beta-tubulin genes of microsporidia. In addition to C. elegans, we found wild C. briggsae and Oscheius tipulae as natural hosts for N. parisii; in addition to C. briggsae, we found C. elegans and C. remanei as natural hosts of N. sp. 1. We further found three new microsporidian species. Two of them are close to the Nematocida species in microsporidian clade II and preliminarily named N.-like sp. 2 and N.-like sp. 3. The third species (or group of species), infecting Oscheius, is very distant, and found in microsporidian clade IV, that includes most human pathogens. They are closest to Orthosomella operophterae (with 18S), and thus preliminarily named Orthosomella-like spp.. Phylogenetic analyses with the 18S and beta-tubulin genes provide the same relationships of Nematocida spp. and the three putative new microsporidia species. Co-infections of two microsporidian species occurr in two wild O. tipulae strains, of which one infected with N. parisii and O.-like spp., the other infected with N. sp. 1 and O.-like spp. (confirmed by specific FISH probes).Secondly, two transgenic C. elegans strains ERT54 jyIs8[C17H1.6p::gfp; myo-2p::mCherry] and ERT72 jyIs15[F26F2.1p::gfp; myo-2::mCherry], which express a constitutive fluorescent Cherry marker and will induce GFP upon infection with N. parisii (Bakowski et al. 2014), were used in infection tests. Our results show that N. parisii, N. sp. 1, N.-like sp. 2 and N.-like sp. 3 can infect ERT54 and ERT72, forming meronts and spores; N. sp. 1 does not induce the GFP reporter (or only rarely), while the other three induce it consistently, suggesting that C. elegans has a different transcriptional response to the infection of N. sp. 1 than to other Nematocida spp. (see also abstract of Luallen et al.). The results also showed that O.-like spp. could not infect the two transgenic C. elegans strains (nor induce the GFP signals).

Jeon, JiHae et al. (2021) International Worm Meeting "N. parisii compensates for genomic loss of dihydroceramide desaturase through reliance on C. elegans sphingolipid biosynthesis"

Reinke, Aaron, Zhao, Winnie, Burton, Nick, Jeon, JiHae

[International Worm Meeting, 2021]

Microsporidia are obligate intracellular parasites that have lost many metabolic enzymes, resulting in the smallest known eukaryotic genomes. Microsporidia are highly dependant upon host nutrients, but it is poorly understood the effect that these parasites have on host metabolism. Using N. parisii, a natural microsporidian parasite of C. elegans, we describe how infection alters host lipid metabolism. We show that the C. elegans lipase ATGL-1 is upregulated in response to N. parisii infection and that host fat stores are depleted. Metabolic profiling of infected animals reveals large changes in lipid composition including changes consistent with nutrient starvation. Additionally, we identify novel ceramides only generated in animals infected with N. parisii. Genomic analysis reveled that N. parisii has lost the enzymes necessary for the de novo synthesis of these sphingolipids. Mutations in C. elegans dihydroceramide desaturases F33D4.4 and ttm-5 or acid ceramidase asah-2, reduce N. parisii growth. Supplementation with sphingosine, a substrate for ceramide synthesis, enhances N. parisii proliferation. Together, our data demonstrate that N. parisii exploits sphingolipid biosynthesis in C. elegans and reveals an evolutionary strategy used by microsporidian parasites to cope with extreme genomic reduction.

di Luccio, Eric et al. (2021) International Worm Meeting "N-NOSE is an innovative, non-invasive and highly sensitive cancer screening method based on the chemotaxis of C. elegans"

Hirotsu, Takaaki, di Luccio, Eric

[International Worm Meeting, 2021]

A simple, affordable, non-invasive, and highly sensitive cancer detection method is the Holy Grail in health screening. Especially early diagnosis of cancer is critical to increase the likelihood of recovery. N-NOSE (Nematode-NOSE) is a pioneering cancer screening method based on the chemotaxis of C. elegans which shows evasive behavior from the urine of healthy individuals while being attracted to the urine of cancer patients. N-NOSE has an average cancer-detection sensitivity of 86.3% in a clinical setting and detect the presence of cancer for 15 types at all stages: stomach, colorectal, lung, breast, uterine, liver, prostate, esophageal, ovarian, bile duct, gallbladder, bladder, kidney, pharyngeal, and pancreatic cancer. N-NOSE relies on a single drop of urine, is non-invasive and cheap, and is notably more sensitive than the blood tumor markers CEA, CA19-9 and CA15-3. In this presentation, we detail the C. elegans olfaction and chemotaxis method behind N-NOSE. Next, we review and summarize our recently published clinical studies on N-NOSE performed in several hospitals in Japan and compare N-NOSE performance to other cancer screening methods. Then, we briefly comment on the next generation of N-NOSE methods currently under development. Lastly, we outline our "World Consortium" R&D grant initiative to anyone in the world with the goal of contributing to the development of next generations of N-NOSE cancer-specific using nematodes. N-NOSE by Hirotsu Bio Science has been launched commercially in 2020 in Japan and is quickly seeking to expand overseas.

Pierce-Shimomura JT et al. (2000) West Coast Worm Meeting "Mutation in the LIM homeobox gene lim-6 disrupts asymmetric function of the ASE chemosensory neurons"

Pearson BJ, Pierce-Shimomura JT, Lockery SR, Gaston MR

[West Coast Worm Meeting, 2000]

C. elegans detects chemicals with 11 pairs of bilaterally symmetric sensory neurons. All of the right-left members of these pairs share similarity in lineage, morphology, and synaptic connectivity. However, one of these pairs (ASE) expresses genes asymmetrically1. ASEL expresses two putative chemoreceptor guanylyl cyclases gcy-6&7 and the homeobox gene lim-6, while ASER expresses a different guanylyl cyclase gcy-5. The ASE neurons are important for chemotaxis to soluble attractants including salts3. We tested whether the asymmetry in expression pattern correlated with an asymmetry in function by ablating ASER, ASEL, or both neurons in individual worms and tracked each animal during chemotaxis in gradients of ammonium chloride, sodium acetate, potassium acetate, and ammonium acetate. To measure chance performance, we tracked worms (n=74) in the absence of a gradient, but analyzed them as if they were in a gradient. Most ASER- (n=29) and ASE- (n=27) worms failed to reach the peak of the NH4Cl gradient, while most ASEL- worms (n=22) reached the peak similar to sham worms (anaesthetized and recovered but not ablated; n=35). Likewise, most ASER- (n=17) and ASE- (n=15) worms failed to reach the peak of the K-acetate gradient, while most ASEL- worms (n=20) reached the peak similar to sham worms. Surprisingly, most ASEL- (n=26) and ASE- (n=12) worms failed to reach the peak of the Na-acetate gradient, while most ASER- worms (n=25) reached the peak similar to sham worms. Worms did not perform better than chance in the ammonium-acetate gradient (n=37). Thus, ASER controls chemotaxis to Cl- and K+, while ASEL controls chemotaxis to Na+. In lim-6 deletion mutants gcy-5 is ectopically expressed in ASEL, while gcy-6&7 expression is correctly restricted to ASEL2. To examine if LIM-6 specifies asymmetry in ASE function, we tested whether ASE neurons retain their asymmetric function in lim-6 mutants. In contrast to wildtype worms, many ASER- lim-6 worms (n=28) were able to reach the peak of the ammonium chloride gradient. Thus, a homeobox gene is involved in breaking symmetry in neuronal function. 1. Yu et al (1997) PNAS 95:3384-7 2. Hobert et al (1999) Dev 126:1547-62 3. Bargmann & Horvitz (1991) Neuron 7:729-42

Jeon M et al. (1997) International C. elegans Meeting "Characterization and Mapping of the Heterochronic Gene lin-42"

Jeon M, Miller EA, Rougvie AE

[International C. elegans Meeting, 1997]

lin-42 is a heterochronic gene for which a single mutant allele has been reported (Liu, 1990). lin-42(n1089) mutants execute seam cell terminal differentiation one stage earlier than wild type animals, during the L3-to-L4 molt. To better characterize the function of lin-42 and understand its role in the heterochronic pathway, we have isolated and characterized seven new lin-42 alleles and are working toward cloning the lin-42 locus. One allele, ve16, is cold sensitive: when raised at 15 C, all animals (n=26) synthesize adult alae during the L3 molt, while at 25 C, no (n=24) L3 molt animals have alae. We are using this allele in temperature-shift experiments to determine the time of lin-42 action with respect to seam cell terminal differentiation. Preliminary results indicate a requirement for lin-42 late during embryogenesis, between egg-laying and hatching, a time prior to the lin-14 temperature sensitive period (Ambros and Horvitz, 1987). In our efforts to clone lin-42, we have isolated two Tc1-tagged lin-42 alleles. We have outcrossed these alleles and have performed Southern blot analysis to identify closely linked Tc1 elements. At present, we have identified five possible linked elements and are assessing the segregation of these elements relative to the lin-42 phenotype. We have also mapped lin-42 between two polymorphic markers, nP48 and veP2. We will perform transformation rescue experiments using YAC clones spanning this region and search for allele-specific RFLPs in DNA isolated from two gamma-ray induced alleles. Our progress in cloning lin-42 using these combined approaches will be reported. Ambros, V. and H.R. Horvitz. (1987). Genes & Dev. 1: 398-414. Liu, Z. (1990). Ph.D. Thesis, Harvard University, Cambridge, MA.

Muimo R et al. (1995) International C. elegans Meeting "NEMATODE ARYLALKYLAMINE N-ACETYL- TRANSFERASES: PURIFICATION AND CHARACTERISATION FROM ASCARIDIA GALLI"

Muimo R, Isaac RE

[International C. elegans Meeting, 1995]

We have previously shown that nematodes have high levels of arylalkylamine N-acetyltransferase activity and that N-acetylation could be a primary route for regulating biogenic amine levels in nematodes. Three arylalkylamine N-acetyltransferase enzymes have recently been identified and purified to electrophoretic homogeneity from a whole worm homogenate preparation of Ascaridia galli using ammonium sulphate precipitation followed by hydrophobic interaction, metal chelate and ion exchange chromatography. A major peak of activity was associated with a 40 kDa protein band on SDS-PAGE and we now report the characterisation of this enzyme. The enzyme activity was very labile and showed a strong dependency on the presence of dithiothreitol for activity during purification. The enzyme had ability to N-acetylate arylalkyl-, phenyl-, and catechol-amines and ,furthermore, the N-acetylation of 5-HT was inhibited in a dose-dependent manner by amines from these groups. Enzyme activity was maximal at pH 6.5 when measured at 37 oC with tyramine and acetyl CoA as substrates. The enzyme had a high affinity for tyramine (Km 6.6 *M) and was inhibited by Cu2+, Zn2+ and Hg2+ions (1mM). The Km for acetyl CoA was 18.8 *M. Arylamines and hydrazines, substrates for the mammalian liver acetyltransferase enzymes, did not inhibit or markedly affect the enzyme activity towards tyramine indicating that the A. galli arylalkylamine N-acetyltransferase enzyme (40 kDa) is not similar to the mammalian liver arylamine N-acetyltransferase enzymes. The other two arylalkylamine N-acetyltransferase enzyme activities are being characterised and their properties will also be reported.

Rahman, Maisha et al. (2021) International Worm Meeting "Specific N-glycans fine-tune somatosensory dendrite patterning"

Diaz-Balzac, Carlos, Bulow, Hannes, Rahman, Maisha

[International Worm Meeting, 2021]

Dendrites are essential for the transmission and processing of stimuli through the nervous system, and their development requires the precise orchestration of many proteins. For example, the C. elegans PVD somatosensory neurons require a conserved cell-adhesion 'menorin' complex - comprised of skin-derived MNR-1/Menorin and SAX-7/L1CAM, muscle secreted LECT-2/Chondromodulin II, and the transmembrane receptor, DMA-1/LRR-TM, in PVD - for their stereotyped arborization. We found that a key enzyme in the N-glycosylation pathway, AMAN-2/Golgi alpha-mannosidase II, plays a role in fine-tuning PVD dendrite patterning. aman-2/GM-II encodes an evolutionarily conserved enzyme required for the formation of complex and paucimannose N-glycans. Mutations in aman-2/GM-II result in dendritic trees with reduced complexity, and enhance the severity of dendrite defects in hypomorphic alleles of members of the menorin complex. AMAN-2/GM-II requires enzymatic activity in PVD to form higher order branches, suggesting that N-glycosylation of a menorin complex component in PVD itself may be significant. Consistent with this hypothesis, we find that DMA-1/LRR-TM is glycosylated in vivo with primarily high-mannose/hybrid N-glycans, and that DMA-1/LRR-TM carries larger, abnormal N-glycans in aman-2/GM-II mutants. Importantly, we determined that the presence of abnormal N-glycans, rather than the absence of wildtype N-glycans, is the root cause of the observed defects, and likely leads to altered protein-protein interactions. Lastly, we found that specific N-glycosylation sites in DMA-1/LRR-TM are important for PVD dendrite morphogenesis. Collectively, our findings suggest that specific N-glycan structures fine-tune dendrite patterning, possibly by regulating complex formation of the DMA-1/LRR receptor with other components of the menorin complex.